http://informahealthcare.com/dct ISSN: 0148-0545 (print), 1525-6014 (electronic) Drug Chem Toxicol, Early Online: 1–6 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/01480545.2014.959174
RESEARCH ARTICLE
Genotoxic evaluation of terbinafine in human lymphocytes in vitro
Drug and Chemical Toxicology Downloaded from informahealthcare.com by York University Libraries on 03/04/15 For personal use only.
Danielle Tolomeotti1, Marialba Avezum Alves de Castro-Prado1, Juliane Rocha de Sant’Anna1, Ana Beatriz Tozzo Martins2, and Valter Augusto Della-Rosa1 1
Departamento de Biotecnologia, Gene´tica e Biologia Celular and 2Departamento de Estatı´stica, Universidade Estadual de Maringa´, Avenida Colombo, Maringa´, Parana´, Brazil Abstract
Keywords
Terbinafine is an antimycotic drug usually used against several superficial fungal infections and with a potential application in the treatment of human cancers. Since to date there are few data on the genotoxic effects of terbinafine in mammalian cells, current study evaluated the potential genotoxic of such antifungal agent in cultured human peripheral blood lymphocytes. Terbinafine was used at the peak plasma concentration (1.0 mg/ml) and in four additional concentrations higher than the human plasmatic peak (5.0 mg/ml, 25.0 mg/ml, 50.0 mg/ml and 100.0 mg/ml). Chromosomal aberrations (CA), sister chromatid exchanges (SCE), micronuclei (MN), nucleoplasmic bridges (NP) and nuclear buds (NB) were scored as genetic endpoints. In all analysis no significant differences (a ¼ 0.05, Kruskal-Wallis test) were observed. Complementary criterion adopted to obtain the final response in cytogenetic agreed with statistical results. Therefore, results of this study showed that terbinafine neither induced CA, SCE, MN, NP and NB nor affected significantly mitotic, replication and cytokinesis-block proliferation indices in any of the tested concentrations. It may be assumed that terbinafine was not genotoxic or cytotoxic to cultured human peripheral blood lymphocytes in our experimental conditions.
Antifungal agent, chromosomal aberrations, micronuclei assay, sister chromatid exchanges
Introduction Fungi are the cause of up to 5% of all infections worldwide. Despite advances in medicine and drug development these infections still remain a significant cause of morbidity and mortality (Sharma & Bhatia, 2011). Terbinafine is a synthetic compound of the allylamine class of antifungals developed in 1983 (Mehta & Bhatt, 2011; Newland & Abdel-Rahman, 2009). It is a powerful specific and selective inhibitor of fungal squalene epoxidase, a key enzyme in the ergosterol biosynthesis pathway (Favre & Ryder, 1996; Petranyi et al., 1987). Terbinafine is among the most common antifungal agents for the treatment of dermatophyte infections of the skin and nails (Newland & Abdel-Rahman, 2009). While the terbinafine antifungal activity has been reported for several human pathogens (Baboota et al., 2007; Baran et al., 2007; Gregurek-Novak, 2004), very few studies have been published on the genotoxicity of this antimycotic drug. In a limited number of toxicological studies, no genotoxic effect of terbinafine was found in human lymphocytes using the sister chromatid exchange test (Bayel, 2006). According to Novartis Pharmaceuticals Corporation, LamisilÕ , terbinafine hydrochloride tablets, in a variety of in vitro genotoxicity Address for correspondence: Marialba Avezum Alves de Castro-Prado, Colombo Avenue, 5790, Maringa, Parana, Brazil, postal code: 87020900. Tel: +55 4430114679. Fax: +55 4430114893. E-mail: [email protected]
History Received 30 June 2014 Accepted 25 August 2014 Published online 18 September 2014
tests, such as mutagenicity in Escherichia coli, Salmonella typhimurium and Chinese hamster fibroblasts and lung cells, gave no evidence of a mutagenic or clastogenic potential (Novartis, 2013). The terbinafine concentrations utilized in these tests, however, were not provided. On the other hand, the in vitro teratogenicity of terbinafine was previously assessed by the whole-embryo culture system in Wistar rats. Although no drug effect on the embryonic development has been observed, terbinafine at a concentration (300 mg/ml) higher than the human peak plasma levels reached in therapy was able to affect the embryonic differentiation. Additionally, a statistically significant increase in the incidence of morphological abnormalities was observed at this high drug concentration (Bechter & Schmid, 1987). In addition to the antimycotic activity of terbinafina, the anti-angiogenic and universal anti-tumor proliferation properties of terbinafine have been recently described. Actually, terbinafine decreased the cell number in cultured human colon adenocarcinoma, hepatocarcinoma and myeloid leukemia cells in a dose-dependent manner (Lee et al., 2003). The anti-proliferation effect of terbinafine was also observed in cultured human oral squamous cell carcinoma (Chien et al., 2012). Therefore, terbinafine is a potential therapeutic drug in the treatment of several human cancers (Chien et al., 2012; Ho et al., 2004; Ho et al., 2006; Lee et al., 2003). Given that multiple properties of terbinafine, including fungicidal, antitumoral and imunostimulatory of
2
D. Tolomeotti et al.
Drug and Chemical Toxicology Downloaded from informahealthcare.com by York University Libraries on 03/04/15 For personal use only.
Figure 1. The chemical structure of terbinafine.
polymorphonuclear leucocytes (Baboota et al., 2007; Baran et al., 2007; Chien et al., 2012; Gregurek-Novak, 2004), and the need for further data on the genotoxicity (DNA fragmentation and DNA damage) of this antimycotic drug, current study investigates the genotoxic effects of terbinafine by employing chromosomal aberrations (CA), sister chromatid exchanges (SCE) and cytokinesis-block micronucleus (CBMN) tests in cultured human lymphocytes.
Materials and Methods Chemicals The test substance terbinafine (CAS no. 78628-80-5), purity (HPLC) 98%, mitomycin C (CAS no. 50-07-7), bromodeoxyuridine (BrdU) (CAS no. 59-14-3), dimethyl sulphoxide (DMSO) (CAS no. 67-68-5), colchicine (CAS no. 64-86-8) and cytochalasin B (CAS no. 14930-96-2) were obtained from Sigma-Aldrich (St.Louis, Mo USA). Other chemicals used for fixation and staining were obtained from Merck. Terbinafine was dissolved in DMSO. The chemical structure of terbinafine (trans-N-(6,6Dimethyl-2-hepten-4ylyl)-N-methyl-1-naphthylmethylamine hydrochloride) is shown in Figure 1. Donors and collection of blood samples The CA test was carried out by using peripheral venous blood samples from four individuals (two males and two females), aged between 19 and 29 years. The SCE and CBMN tests were carried out by using blood samples from two individuals (one male and one female, for each test), aged 25 and 19 and 29 and 19 years, respectively. The study was approved by the local Ethics Committee. All volunteers gave their informed and free consent to participate in the study and filed the required forms. Acceptability criteria to ensure reliability of the experiment were: no intake of alcohol and drugs, no smoking, no exposure to ionizing radiation within the previous 6 months, no history of recent viral infection and no medical therapy. Peripheral venous blood was collected and transferred to tubes containing sodium heparin as anticoagulant and processed within 1 h after collection. Whole blood was centrifuged at 1000 rpm and buffy coatrich plasma was used for cultures. Lymphocyte cultures and treatments Buffy coat-rich plasm (0.5 mL) was added to 4.5 mL culture medium containing 82% RPMI 1640 medium, 15% fetal calf serum, 2% phytohemagglutinin to stimulate cell division and 1% L-glutamine. All compounds were obtained from Gibco. Human lymphocyte cultures were incubated at 37 C for 72 h. The concentrations of terbinafine tested in the present study ranged from 1.0 to 100.0 mg/ml. At this point, it must be
Drug Chem Toxicol, Early Online: 1–6
mentioned that the concentration of 1 mg/ml, the lower tested in the present study, represent the peak plasma concentration of this drug following oral administration in humans (Kovarik et al., 1995). The cultures were treated with terbinafine at concentrations 1.0 mg/ml, 5.0 mg/ml, 25.0 mg/ml, 50.0 mg/ml and 100.0 mg/ml. An untreated culture, a solvent control (DMSO, 10.0 ml/ml and 20.0 ml/ml), a positive control (mitomycin C, 0.1 mg/ml) and five cultures treated with different concentrations of terbinafine were applied to each donor. All cultures were maintained under identical conditions. Chromosomal aberrations and sister chromatid exchanges tests For CA and SCE studies, metaphase preparations were obtained as described by Moorhead et al.(1960) with modifications. For SCE test, the culture medium was supplemented with 10 mg/ml BrdU and cultures were wrapped in aluminum foil to avoid exposure to light. Terbinafine was added 48 h after the initiation of culture. Colchicine (4.105 M) was added 2 h before the collection of the cultures to obtain metaphases. The cultures were treated with a hypotonic solution (0.075 M KCI) for 20 min at 37 C and then fixed twice with cold methanol:acetic acid (3:1, v/v). Three drops of the fixed cell suspension were dropped on clean and cold slides which were maintained at 21 C for 3 days. For CA test, slides were incubated in 2 x SSC (0.3 M sodium chloride and 0.03 M trisodium citrate) solution at 60 C for 5 min and stained with 2% Giemsa solution prepared in a So¨rensen buffer (pH 6.8) for 5 min, washed in distilled water and dried at room temperature. For SCE test, the slides were stained according to fluorescence plus Giemsa technique (Korenberg & Freedlender, 1974), with modifications. In the two tests, all slides were coded prior to scoring and analyzed by a single cytogeneticist. In the CA assay, the frequency of the cells with structural and numeric CA was scored in 100 well-spread metaphases, containing 46 ± 1 chromosomes, per culture, for each treatment and donor (a total of 400 metaphases). Aberrations were classified according to the recommendations of the International System for Human Cytogenetic Nomenclature (ISCN, 2013), as follows: chromatid and chromosome breaks, acentric chromosome fragments, chromatid and chromosome exchanges. Achromatic lesions smaller than the width of a chromatid and continuous with the chromosome axis were recorded into: chromatid and chromosome gaps, albeit not considered as CA. Among the numeric CA observed, only polyploidy was taken into account. The number of each aberration type was summarized for each treatment and the count of cells with CA and the number of CA per cell (CA/cell) were expressed as the mean number ± standard error (SE). The mitotic index (MI) was calculated as the proportion of metaphases in 1000 cells analyzed per culture, for each treatment and donor (a total of 4000 cells) and expressed as mean percentage ± SE. In the SCE test, 25 well-spread second division metaphases, all with 46 chromosomes, were analyzed per culture, for each treatment and donor (a total of 50 metaphases). SCE count was performed according to Lambert et al. (1976), but centromeric exchanges were not considered. The results for
Genotoxic potential of terbinafine
DOI: 10.3109/01480545.2014.959174
Statistical analysis and interpretation of the results
each treatment were expressed as the mean number of SCE per cell (SCE/cell) ± SE. Further, for the determination of the replication index (RI), the proportion of first (M1), second (M2) and third (M3) division cells was scored in 100 consecutive metaphases per culture, for each treatment and donor (a total of 200 cells) and was calculated by the formula: RI ¼ M1+2M2+3M3/100, and expressed as the mean number ± SE.
Statistical analysis was performed by Kruskal-Wallis test, comparing the experimental rates of the tested concentrations of terbinafine and the solvent control obtained in CA, SCE and CBMN tests. Dose–response relationships were determined from the correlation coefficients (r), considered as strong to perfect correlation the following values: 0.75 5 r 51.00 or 1 5 r 5 0.75 (perfect if r ¼ 1 or r ¼ 1) (Vieira, 2008). For all analyses, a significance level of 5% (a ¼ 0.05) was used. For interpretation of results beyond the statistical analysis, complementary criteria were also considered as a positive response: an induction of 45% of cells with CA (Parry et al., 2010), for CA test, and an increase in the frequency of SCE in tested concentrations to two times when compared to the solvent control (Varella-Garcia, 1991), for SCE test. In CBMN assay, the MN, NP and NB rates suggested by Fenech (2007) were considered as indicative of spontaneous induction.
Cytokinesis-block micronucleus test
Drug and Chemical Toxicology Downloaded from informahealthcare.com by York University Libraries on 03/04/15 For personal use only.
3
The CBMN test was carried and analyzed as described by Fenech (2007), with modifications. Terbinafine was added 24 h after the initiation of culture. Cytochalasin B (6 mg/ml) was added 28 h before the collection of the cultures to arrest cytokinesis and obtain binucleated (BN) cells. The cultures were treated with hypotonic solution (0.075 M KCl) at 4 C for 5 min and then fixed twice with cold methanol:acetic acid (3:1, v/v). In the last fixative, 1% formaldehyde was added to preserve the cytoplasm. Slides were prepared by dropping three drops of the fixed cell suspension on clean and cold slides and then air-dried. The slides were incubated at 21 C for 3 days and stained with 5% Giemsa solution prepared in a So¨rensen buffer (pH 6.8), for 5 min, washed in distilled water and dried at room temperature. All slides were coded prior to scoring and analyzed by a single cytogeneticist. Frequency of micronuclei (MN), nucleoplasmic bridges (NP) and nuclear buds (NB) were scored in the CBMN assay. A total of 1000 BN lymphocytes were analyzed per culture, for each treatment and donor (a total of 2000 cells) to determine the number of MN, NP and NB. Total number of MN, NP and NB scored were summarized for each treatment and the count of BN cells with MN and NB was expressed as the mean number ± SE. For determining cytokinesis-block proliferation index (CBPI), 500 cells with well-preserved cytoplasm, containing 1–4 nuclei, were scored per culture, for each treatment and donor. CBPI was calculated according to Surralle´s et al. (1995) using the following formula: N1 þ2N2 þ3ðN3 þN4 Þ CBPI ¼ N
Results Five different concentrations (1.0 mg/ml, 5.0 mg/ml, 25.0 mg/ml, 50.0 mg/ml and 100.0 mg/ml) and eight different parameters (CA, SCE, MN, NP, NB, MI, RI and CBPI) were evaluated to determine the genotoxic and cytotoxic/cytostatic effects of terbinafine in cultured human peripheral lymphocytes. The results of CA test are shown in Table 1.Three types of structural CA were observed for terbinafine in tested concentrations: chromosome and chromatid breaks and acentric chromosome fragments, giving a predominance of chromatid breaks. Other structural aberrations as dicentric and ring chromosomes and chromosomal rearrangements were not observed in any of the tested concentrations of terbinafine. Polyploidy was observed. Among the tested concentrations of terbinafine and solvent control, no significant differences regarding the numbers the cells with CA (p ¼ 0.1943), CA/cell (p ¼ 0.2065) and percentages of MI (p ¼ 0.8003) were reported. Further, the number of cells with CA was not increased in a dose-dependent manner (r ¼ 0.69). No more than 5% of cells with CA, in each one of tested concentrations of terbinafine, were observed, the maximum rate was 4.0 ± 1.29% (100.0 mg/ml). The effects of terbinafine on the SCE and RI are summarized in Table 2. There was no significant difference
where, N1–N4 represent the number of cells with 1–4 nuclei, respectively, and N is the total number of cells scored and expressed as the mean number ± SE.
Table 1. Chromosomal aberrations and mitotic index in human lymphocytes treated with terbinafine in vitro. Treatment Test substance Untreated culture Solvent control Positive control (mitomycin C) Terbinafine
Chromosomal aberrations
Period (h)
Dose
csb
ctb
f
cte
p
CCA (mean* ± SE)
CA/cell (mean ± SE)
MI(%) (mean ± SE)
– 48 48 48
– 20.0 ml/ml 0.1mg/ml 1.0 mg/ml 5.0 mg/ml 25.0 mg/ml 50.0 mg/ml 100.0 mg/ml
3 1 165 0 2 8 3 4
2 10 83 2 6 5 4 11
0 0 9 1 1 1 2 1
0 0 18 0 0 0 0 0
0 0 0 2 0 1 0 2
1.25 ± 0.48 2.50 ± 1.55 38.75 ± 14.48 1.25 ± 0.63 2.25 ± 0.25 3.75 ± 1.18 2.25 ± 0.95 4.00 ± 1.29
0.0125 ± 0.005 0.0275 ± 0.018 0.6875 ± 0.340 0.0125 ± 0.006 0.0225 ± 0.002 0.0375 ± 0.012 0.0225 ± 0.009 0.0450 ± 0.017
3.525 ± 0.14 2.750 ± 0.53 0.875 ± 0.24 2.700 ± 0.38 2.575 ± 0.53 2.325 ± 0.33 2.500 ± 0.33 2.125 ± 0.26
csb, chromosome break; ctb, chromatid break; f, acentric chromosome fragment; cte, chromatid exchange; p, polyploidy; CCA, cells with chromosomal aberrations; CA/cell, chromosomal aberrations per cell; MI, mitotic index. Four hundred metaphases were scored for each treatment to analyze chromosomal aberrations and 4000 lymphocytes were scored for each dose level for the MI. *The average sample rate coincides with CCA percentage.
4
D. Tolomeotti et al.
Drug Chem Toxicol, Early Online: 1–6
Table 2. Sister chromatid exchanges and replication index in human lymphocytes treated with terbinafine in vitro. Test substance
Treatment Period (h)
Untreated culture Solvent control Positive control (mitomycin C) Terbinafine
Min-max
SCE/cell (mean ± SE)
RI (mean ± SE)
1–10 1–15 6–41 0–14 1–14 2–18 2–14 1–22
5.52 ± 0.36 6.32 ± 1.92 24.42 ± 4.06 6.60 ± 2.12 6.14 ± 0.98 6.64 ± 1.32 6.66 ± 0.46 6.68 ± 1.36
2.435 ± 0.055 2.195 ± 0.165 1.745 ± 0.015 2.265 ± 0.135 2.210 ± 0.240 2.145 ± 0.085 2.355 ± 0.025 1.995 ± 0.085
Dose
– 48 48 48
– 20.0 ml/ml 0.1 mg/ml 1.0 mg/ml 5.0 mg/ml 25.0 mg/ml 50.0 mg/ml 100.0 mg/ml
Drug and Chemical Toxicology Downloaded from informahealthcare.com by York University Libraries on 03/04/15 For personal use only.
SCE/cell, sister chromatid exchanges per cell; RI, replication index. Fifty metaphases were scored for each dose level in the SCE test. Two hundred metaphases were scored in each dose level for RI.
Table 3. Micronuclei, nuclear buds and cytokinesis-block proliferation index (CBPI) in human lymphocytes treated with terbinafine in vitro. Treatment Test substance Negative control Solvent control Positive control Terbinafine
Period (h)
Concentration
BN scored
MN per 1000 BN (mean ± SE)
Total number of MN
BNNB (mean ± SE)
Total number of buds
CBPI (mean ± SE)
24 24 24 24 24 24 24 24
– 10 mL/mL 0.1 mg/mL 1 mg/mL 5 mg/mL 25 mg/mL 50 mg/mL 100 mg/mL
2000 2000 2000 2000 2000 2000 2000 2000
6.5 ± 0.50 6.0 ± 2.01 96.5 ± 18.56 7.5 ± 0.50 8.5 ± 0.50 10.0 ± 1.00 10.5 ± 0.50 11.5 ± 1.50
13 12 193 15 17 20 21 23
2.0 ± 0 3.5 ± 0.50 10.0 ± 0 3.0 ± 1.003 4.0 ± 0 1.0 ± 1.003 4.5 ± 0.50 5.5 ± 0.50
4 7 20 6 8 2 9 11
2.01 ± 0.01 2.01 ± 0.01 1.9 ± 0.21 2.0 ± 0.13 2.0 ± 0.21 1.9 ± 0.06 1.7 ± 0.12 1.43 ± 0.18
BN: binucleated cells; MN: micronuclei; BNNB: binucleated cells with nuclear buds; NB: nuclear buds; CBPI: cytokinesis-block proliferation index. Positive control: mitomicyn C.
in the number of SCE/cell (p ¼ 0.9946) and RI (p ¼ 0.5945) among the tested concentrations of terbinafine and solvent control. The number of SCE/cell did not increase (r ¼ 0.54) neither RI decrease (r ¼ 0.59) in a dose-dependent manner. No differences were observed regarding the frequency of the SCE/cell when compared to solvent control (6.32 ± 1.92), with maximum rate 6.68 ± 1.36 (100.0 mg/ml). Table 3 shows the results obtained in the CBMN assay. Among the tested concentrations of terbinafine and solvent control, there was no significant difference in the number of BN cells with MN (p ¼ 0,1103) and NB (p ¼ 0,03587). No significant differences were found in CBPI (p ¼ 0,1853) when treatment and solvent control are compared. NP were not observed in any of the tested terbinafine concentrations.
Discussion Terbinafine is an orally and topically active drug with fungicidal activity against a broad spectrum of fungi (Kovaric et al., 1995). Studies showed that terbinafine has anti-angiogenic and universal anti-tumor proliferation properties, which makes it a very attractive agent for cancer therapy (Ho et al., 2004, 2006; Lee et al., 2003). Chien et al. (2012) demonstrated for the first time that terbinafine might be a potential therapeutic tool in the treatment of oral cancer. Genetic toxicology studies play an important role in evaluating health hazards associated with the exposure of humans and living organisms to chemical substances (Ohno et al., 2005). In general, CA, SCE and MN are considered to
be essential markers of genotoxicity in vitro studies (Celik & Eke, 2011). Current study investigates for the first time the genotoxic potential of terbinafine using CA, SCE and CBMN tests in cultured human lymphocytes. Five terbinafine concentrations were evaluated in our study: the peak plasma concentration of terbinafine following oral administration in humans (Kovaric et al., 1995) (1.0 mg/ml) and four additional concentrations higher than the plasma concentration (5.0 mg/ml, 25.0 mg/ml, 50.0 mg/ml and 100.0 mg/ml). No significant genotoxic effects were observed by the five different parameters (CA, SCE, MN, NP and NB) in all terbinafine tested concentrations. Furthermore, the results were also negative in terms of cytotoxicity and cytostasis, since no significant differences were found in MI, RI and CBPI for any terbinafine concentration. The CA assay in cultured cells has been widely used for many years, and it has been proved to be an useful and sensitive test for the detection of genotoxic agents (Rao et al., 2004). The varieties of CA observed in human lymphocytes treated in vitro with terbinafine were observed for fluconazole, another antifungal drug (Yu¨zba¸siog˘lu et al., 2008). Although in our analysis the structural and numeric CA has also been observed, this datum was not relevant. The limit value of cells with CA, based on Parry et al. (2010), was not exceeded in untreated culture, solvent control or any of the terbinafine concentrations. This complementary criterion agrees with statistical results. In positive control, 38.75 ± 14.48% of the cells with CA, were observed. This datum indicates that this criterion is strong enough to obtain the final response in CA test. The frequencies of CA/cell in untreated
Genotoxic potential of terbinafine
Drug and Chemical Toxicology Downloaded from informahealthcare.com by York University Libraries on 03/04/15 For personal use only.
DOI: 10.3109/01480545.2014.959174
culture, solvent control and tested concentrations of terbinafine ranged from 1.25 to 4.5%, which is in conformity with the proposed by Kasˇuba et al. (1995) for spontaneous CA (range 0 to 6.7%). SCE test is a sensitive test for monitoring DNA damage (Pongsavee, 2009) although little is known about its molecular basis. This analysis has been used to investigate the genotoxic effects of several chemical agents, for example, food additives (Yilmaz et al., 2008, 2009; Zengin et al., 2011). SCE occurs in human lymphocyte cultures, with or without induction by physical or chemical agents, and it seems to reflect the DNA repair by homologous recombination (Garcia-Sagredo, 2008). The frequency of the SCE/cell increases after exposure of cells to genotoxic agents, as demonstrated by Aydemir et al. (2005), C¸elik et al. (2010) and Mamur et al. (2010). The frequencies of SCE/cell in untreated culture, solvent control and all terbinafine concentrations ranged from 5.52 ± 0.36–6.68 ± 1.36 SCE/cell, which is in accordance with the basal frequency suggested by Natarajan & Obe (1986), within the 5–10 SCE/cell. Our results showed that terbinafine does not induce SCE in human lymphocytes because, beyond the statistical results, in none of the tested concentration of terbinafine was observed the two-fold increase in SCE over that of the solvent control. In positive control, 24.42 ± 4.06 SCE/cell was observed, approximately four times greater than that in solvent control. This datum demonstrates the validity of this complementary criterion adopted to obtain the final response in the SCE test. The in vitro MN assay is currently being considered as a suitable method for testing the genotoxicity of chemical and pharmaceutical compounds (Fenech, 2000). The MN, NP and NB rates suggested by Fenech (2007), adopted in this study as indicative of spontaneous induction, were not exceeded in untreated culture, solvent control and terbinafine group. This complementary criterion agrees with statistical results. Results indicate that, under current test conditions, terbinafine does not induce chromosome breaks and/or gain or loss in human lymphocytes. Similar results were found for other drugs (Ila & Ilhan, 2012; Kasurka et al., 2011). However, griseofulvin, another antifungal drug, induced MN formation in cultured human lymphocytes and was considered a strong aneuploidogenic agent (Kolachana & Smith, 1994). MI explains the effects of the chemical compounds on the G2 stage of the cell cycle, while RI reflects the effects on the S and G2 stages (Arslan et al., 2008). CBPI estimates the average number of cell division of a cell population and may be considered a cell kinetic index (Surralle´s et al., 1995). Since a significant reduction of MI, RI and CBPI rates in cultures treated with terbinafine was not observed, terbinafine does not seem to produce effects on the proliferation/mitotic index when its concentration is equal to or less than 100.0 mg/ ml. Terbinafine at concentrations 30–60 mM did not inhibit the growth rate of the cultured human gingival fibroblasts, and at doses 10–150 mM it was not cytotoxic for the cultured untransformed human fibroblasts, with no effect on cell proliferation in this culture (Lee et al., 2003). Therefore, the data obtained in current study seem to corroborate the results obtained by Lee et al. (2003).
5
Conclusion The results in the current study show that terbinafine does not induce clastogenesis, DNA effects, aneugenesis, cytotoxicity and cytostasis in human lymphocytes in vitro. Since very few studies have been published on the genotoxicity of this antimicotic drug, our results contribute with data towards a safer use of the drug, already widely used in the treatment of dermatomycoses in humans and with a potential application as an anti-angiogenic and anti-tumor agent.
Acknowledgements We are grateful to Fundac¸a˜o Arauca´ria de Apoio ao Desenvolvimento Cientı´fico e Tecnolo´gico do Parana´ for financial support. Tolomeotti, D. was the recipient of a Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) fellowship. Sant’Anna, J.R. is the recipient of a Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES) fellowship.
Declaration of interest The authors declare that there is no conflict of interest.
References Arslan M, Topaktas M, Rencuzogullari E. (2008). The effects of boric acid on sister chromatid exchanges and chromosome aberrations in cultured human lymphocytes. Cytotechnology, 56:91–96. Aydemir N, C¸elikler S, Bilalog˘lu R. (2005). In vitro genotoxic effects of the anticancer drug gemcitabine in human lymphocytes. Mutat Res, 582:35–41. Baboota S, Al-Azaki A, Kohli K, et al. (2007). Development and evaluation of a microemulsion formulation for transdermal delivery of terbinafine. PDA J Pharmaceut Sci Technol, 61:276–285. Baran R, Sigurgeirsson B, de Berker D, et al. (2007). A multicentre, randomized, controlled study of the efficacy, safety and costeffectiveness of a combination therapy with amorolfine nail lacquer and oral terbinafine compared with oral terbinafine alone for the treatment of onychomycosis with matrix involvement. British J Dermatol, 157:149–157. Bayel I. (2006). Terbinafin’in insan lenfosit ku¨ltu¨rlerindeki etkilerinin karde¸s kromatid degˇi¸simi (sister chromatid exchange) yo¨ntemi ile in ¨ niversitesi vitro ara¸stırılması. (Yu¨ksek Lisans Tezi). Mersin: Mersin U Sag_lik Bilimleri Enstitu¨su¨,61s. Bechter R, Schmid BP. (1987). Teratogenicity in vitro - A comparative study of four antimycotic drugs using the whole-embryo culture system. Toxicol In Vitro, 1:11–15. C¸elik A, Eke D. (2011). The assessment of cytotoxicity and genotoxicity of tetracycline antibiotic in human blood lymphocytes using CBMN and SCE analysis, in vitro. Int J Hum Genet, 11:23–29. Celik M, Aksoy H, Yilmaz S. (2010). Evaluation of beauvericin genotoxicity with the chromosomal aberrations, sister-chromatid exchanges and micronucleus assays. Ecotoxicol Environ. Saf, 73:1553–1557. Chien MH, Lee TS, Kao C, et al. (2012). Terbinafine inhibits oral squamous cell carcinoma growth through anti-cancer cell proliferation and anti-angiogenesis. Mol Carcinog, 51:389–399. Favre B, Ryder NS. (1996). Characterization of squalene epoxidase activity from the dermatophyte Trichophyton rubrum and its inhibition by terbinafine and other antimycotic agents. Antimicrob Agents Chemother, 40:443–447. Fenech M. (2000). A mathematical model of the in vitro micronucleus assay predicts false negative results if micronuclei are not specifically scored in binucleated cells or in cells that have completed one nuclear division. Mutagenesis, 15:329–336. Fenech M. (2007). Cytokinesis-block micronucleus cytome assay. Nat Protoc, 2:1084–1104.
Drug and Chemical Toxicology Downloaded from informahealthcare.com by York University Libraries on 03/04/15 For personal use only.
6
D. Tolomeotti et al.
Garcia-Sagredo JM. (2008). Fifty years of cytogenetics: a parallel view of the evolution of cytogenetics and genotoxicology. Biochim Biophys Acta, 1779:363–375. Gregurek-Novak T. (2004). Effect of infection with Trichophyton mentagrophytes varietas interdigitale on phagocytosis in humans. J European Acad Dermatol Venereol, 18:160–163. Ho PY, Liang YC, Ho YS, et al. (2004). Inhibition of human vascular endothelial cells proliferation by terbinafine. Int J Cancer, 111:51–59. Ho PY, Zhong WB, Ho YS, Lee WS. (2006). Terbinafine inhibits endothelial cell migration through suppression of the Rho-mediated pathway. Mol Cancer Ther, 5:3130–3138. Ila HB, Ilhan A. (2012). In vitro genotoxic perspective of Tamiflu. Cytotechnology, 64:443–449. ISCN (2013). An international system for human cytogenetic nomenclature (2013). In: Shaffer LG, McGowan-Jordan JR, Schmid M, eds. (Karger Medical and Scientific Publishers). Kasˇuba V, Sˇentija K, Garaj-Vrhovac V, Fucˇic´ A. (1995). Chromosome aberrations in peripheral blood lymphocytes from control individuals. Mutat Res, 346:187–193. Kasurka CB, S ¸ ekerog˘lu ZA, S ¸ ekerog˘lu V. (2011). Evaluation of the genotoxicity and cytotoxicity of fexofenadine in cultured human peripheral blood lymphocytes. Toxicol In Vitro, 25:1480–1484. Kolachana P, Smith MT. (1994). Induction of kinetochore-positive micronuclei in human lymphocytes by the anti-fungal drug griseofulvin. Mutat Res, 322:151–159. Korenberg JR, Freedlender EF. (1974). Giemsa technique for the detection of sister chromatid exchanges. Chromosoma, 48:355–360. Kovarik JM, Mueller EA, Zehender H, et al. (1995). Multiple-dose pharmacokinetics and distribution in tissue of terbinafine and metabolites. Antimicrob Agents Chemother, 39:2738–2741. Lambert BO, Hansson K, Lindsten J, et al. (1976). Bromodeoxyuridineinduced sister chromatid exchanges in human lymphocytes. Hereditas, 83:163–174. Lee WS, Chen RJ, Wang YJ, et al. (2003). In vitro and in vivo studies of the anticancer action of terbinafine in human cancer cell lines: G0/G1 p53-associated cell cycle arrest. Int J Cancer, 106:125–137. ¨ nal F, Yilmaz S. (2010). Does potassium Mamur S, Yu¨zba¸siog˘lu D, U sorbate induce genotoxic or mutagenic effects in lymphocytes? Toxicol In Vitro, 24:790–794 Mehta K, Bhatt DC. (2011). Preparation, optimization and in vitro microbiological efficacy of antifungal microemulsion. Int J Pharmaceut Sci Res, 2:2424–2429. Moorhead PS, Nowell PC, Mellman WJ, et al. (1960). Chromosome preparations of leukocytes cultured from human peripheral blood. Exp Cell Res 1960, 20:613–616.
Drug Chem Toxicol, Early Online: 1–6
Natarajan AT, Obe G. (1986). How do in vivo mammalian assays compare to in vitro assays in their ability to detect mutagens? Mutat Res, 167:189–201. Newland JG, Abdel-Rahman SM. (2009). Update on terbinafine with a focus on dermatophytoses. Clin Cosmet Investig Dermatol, 2:49–63. Novartis (2013). LamisilÕ (terbinafine hydrochloride). Avaliable at: http://www.acessdata.fda.gov/drugsatfda_docs/label/2013/ 022071s009Ibl.pdf. Ohno K, Tanaka-Azuma Y, Yoneda Y, Yamada T. (2005). Genotoxicity test system based on p53R2 gene expression in human cells: examination with 80 chemicals. Mutat Res, 588:47–57. Parry JM, Parry E, Phrakonkham P, Corvi R. (2010). Analysis of published data for top concentration considerations in mammalian cell genotoxicity testing. Mutagenesis, 25:531–538. Petranyi G, Meingassner JG, Mieth H. (1987). Antifungal activity of the allylamine derivative terbinafine in vitro. Antimicrob Agents Chemother, 31:1365–1368. Pongsavee M. (2009). Effect of borax on immune cell proliferation and sister chromatid exchange in human chromosomes. J Occup Med Toxicol, 4:1–6. Rao KS, Xu Y, Shaw E, Parton JW. (2004). Mutagenicity testing applied for regulation of developing products. Curr Separations, 20:141–144. Sharma V, Bhatia R. (2011). Triazoles in antifungal therapy: A review. Int J Pharm Biomed Res, 2:417–427. Surralle´s J, Xamena N, Creus A, et al. (1995). Induction of micronuclei by five pyrethroid insecticides in whole blood and isolated human lymphocytes cultures. Mutat Res, 341:169–184. Varella-Garcia M. (1991). Teste de trocas entre croma´tides-irma˜s. In: Rabello-Gay MN, Rodrigues MA, Monteleone-Neto R, editors. Mutageˆnese, teratogeˆnese e carcinogeˆnese: me´todos e crite´rios de avaliac¸a˜o. Ribeira˜o Preto, SP: SBG, p. 123–139. Vieira S. (2008). Introduc¸a˜o a` bioestatı´stica. Rio de Janeiro, RJ, Brasil: Elsevier. ¨ nal F, Yu¨zba¸siog˘lu D, Aksoy H. (2008). Clastogenic effects Yilmaz S, U of food additive citric acid in human peripheral lymphocytes. Cytotechnology, 56:137–144. ¨ nal F, Yu¨zba¸siog˘lu D. (2009). The in vitro genotoxicity Yilmaz S, U of benzoic acid in human peripheral blood lymphocytes. Cytotechnology, 60:55–61. ¨ nal F, Yilmaz S, et al. (2008). Genotoxicity testing of Yu¨zba¸siog˘lu D, U fluconazole in vivo and in vitro. Mutat Res, 649:155–160. ¨ nal F, et al. (2011). The evaluation of the Zengin N, Yu¨zba¸siog˘lu D, U genotoxicity of two food preservatives: Sodium benzoate and potassium benzoate. Food Chem Toxicol, 49:763–769.
RESEARCH ARTICLE
Genotoxic evaluation of terbinafine in human lymphocytes in vitro
Drug and Chemical Toxicology Downloaded from informahealthcare.com by York University Libraries on 03/04/15 For personal use only.
Danielle Tolomeotti1, Marialba Avezum Alves de Castro-Prado1, Juliane Rocha de Sant’Anna1, Ana Beatriz Tozzo Martins2, and Valter Augusto Della-Rosa1 1
Departamento de Biotecnologia, Gene´tica e Biologia Celular and 2Departamento de Estatı´stica, Universidade Estadual de Maringa´, Avenida Colombo, Maringa´, Parana´, Brazil Abstract
Keywords
Terbinafine is an antimycotic drug usually used against several superficial fungal infections and with a potential application in the treatment of human cancers. Since to date there are few data on the genotoxic effects of terbinafine in mammalian cells, current study evaluated the potential genotoxic of such antifungal agent in cultured human peripheral blood lymphocytes. Terbinafine was used at the peak plasma concentration (1.0 mg/ml) and in four additional concentrations higher than the human plasmatic peak (5.0 mg/ml, 25.0 mg/ml, 50.0 mg/ml and 100.0 mg/ml). Chromosomal aberrations (CA), sister chromatid exchanges (SCE), micronuclei (MN), nucleoplasmic bridges (NP) and nuclear buds (NB) were scored as genetic endpoints. In all analysis no significant differences (a ¼ 0.05, Kruskal-Wallis test) were observed. Complementary criterion adopted to obtain the final response in cytogenetic agreed with statistical results. Therefore, results of this study showed that terbinafine neither induced CA, SCE, MN, NP and NB nor affected significantly mitotic, replication and cytokinesis-block proliferation indices in any of the tested concentrations. It may be assumed that terbinafine was not genotoxic or cytotoxic to cultured human peripheral blood lymphocytes in our experimental conditions.
Antifungal agent, chromosomal aberrations, micronuclei assay, sister chromatid exchanges
Introduction Fungi are the cause of up to 5% of all infections worldwide. Despite advances in medicine and drug development these infections still remain a significant cause of morbidity and mortality (Sharma & Bhatia, 2011). Terbinafine is a synthetic compound of the allylamine class of antifungals developed in 1983 (Mehta & Bhatt, 2011; Newland & Abdel-Rahman, 2009). It is a powerful specific and selective inhibitor of fungal squalene epoxidase, a key enzyme in the ergosterol biosynthesis pathway (Favre & Ryder, 1996; Petranyi et al., 1987). Terbinafine is among the most common antifungal agents for the treatment of dermatophyte infections of the skin and nails (Newland & Abdel-Rahman, 2009). While the terbinafine antifungal activity has been reported for several human pathogens (Baboota et al., 2007; Baran et al., 2007; Gregurek-Novak, 2004), very few studies have been published on the genotoxicity of this antimycotic drug. In a limited number of toxicological studies, no genotoxic effect of terbinafine was found in human lymphocytes using the sister chromatid exchange test (Bayel, 2006). According to Novartis Pharmaceuticals Corporation, LamisilÕ , terbinafine hydrochloride tablets, in a variety of in vitro genotoxicity Address for correspondence: Marialba Avezum Alves de Castro-Prado, Colombo Avenue, 5790, Maringa, Parana, Brazil, postal code: 87020900. Tel: +55 4430114679. Fax: +55 4430114893. E-mail: [email protected]
History Received 30 June 2014 Accepted 25 August 2014 Published online 18 September 2014
tests, such as mutagenicity in Escherichia coli, Salmonella typhimurium and Chinese hamster fibroblasts and lung cells, gave no evidence of a mutagenic or clastogenic potential (Novartis, 2013). The terbinafine concentrations utilized in these tests, however, were not provided. On the other hand, the in vitro teratogenicity of terbinafine was previously assessed by the whole-embryo culture system in Wistar rats. Although no drug effect on the embryonic development has been observed, terbinafine at a concentration (300 mg/ml) higher than the human peak plasma levels reached in therapy was able to affect the embryonic differentiation. Additionally, a statistically significant increase in the incidence of morphological abnormalities was observed at this high drug concentration (Bechter & Schmid, 1987). In addition to the antimycotic activity of terbinafina, the anti-angiogenic and universal anti-tumor proliferation properties of terbinafine have been recently described. Actually, terbinafine decreased the cell number in cultured human colon adenocarcinoma, hepatocarcinoma and myeloid leukemia cells in a dose-dependent manner (Lee et al., 2003). The anti-proliferation effect of terbinafine was also observed in cultured human oral squamous cell carcinoma (Chien et al., 2012). Therefore, terbinafine is a potential therapeutic drug in the treatment of several human cancers (Chien et al., 2012; Ho et al., 2004; Ho et al., 2006; Lee et al., 2003). Given that multiple properties of terbinafine, including fungicidal, antitumoral and imunostimulatory of
2
D. Tolomeotti et al.
Drug and Chemical Toxicology Downloaded from informahealthcare.com by York University Libraries on 03/04/15 For personal use only.
Figure 1. The chemical structure of terbinafine.
polymorphonuclear leucocytes (Baboota et al., 2007; Baran et al., 2007; Chien et al., 2012; Gregurek-Novak, 2004), and the need for further data on the genotoxicity (DNA fragmentation and DNA damage) of this antimycotic drug, current study investigates the genotoxic effects of terbinafine by employing chromosomal aberrations (CA), sister chromatid exchanges (SCE) and cytokinesis-block micronucleus (CBMN) tests in cultured human lymphocytes.
Materials and Methods Chemicals The test substance terbinafine (CAS no. 78628-80-5), purity (HPLC) 98%, mitomycin C (CAS no. 50-07-7), bromodeoxyuridine (BrdU) (CAS no. 59-14-3), dimethyl sulphoxide (DMSO) (CAS no. 67-68-5), colchicine (CAS no. 64-86-8) and cytochalasin B (CAS no. 14930-96-2) were obtained from Sigma-Aldrich (St.Louis, Mo USA). Other chemicals used for fixation and staining were obtained from Merck. Terbinafine was dissolved in DMSO. The chemical structure of terbinafine (trans-N-(6,6Dimethyl-2-hepten-4ylyl)-N-methyl-1-naphthylmethylamine hydrochloride) is shown in Figure 1. Donors and collection of blood samples The CA test was carried out by using peripheral venous blood samples from four individuals (two males and two females), aged between 19 and 29 years. The SCE and CBMN tests were carried out by using blood samples from two individuals (one male and one female, for each test), aged 25 and 19 and 29 and 19 years, respectively. The study was approved by the local Ethics Committee. All volunteers gave their informed and free consent to participate in the study and filed the required forms. Acceptability criteria to ensure reliability of the experiment were: no intake of alcohol and drugs, no smoking, no exposure to ionizing radiation within the previous 6 months, no history of recent viral infection and no medical therapy. Peripheral venous blood was collected and transferred to tubes containing sodium heparin as anticoagulant and processed within 1 h after collection. Whole blood was centrifuged at 1000 rpm and buffy coatrich plasma was used for cultures. Lymphocyte cultures and treatments Buffy coat-rich plasm (0.5 mL) was added to 4.5 mL culture medium containing 82% RPMI 1640 medium, 15% fetal calf serum, 2% phytohemagglutinin to stimulate cell division and 1% L-glutamine. All compounds were obtained from Gibco. Human lymphocyte cultures were incubated at 37 C for 72 h. The concentrations of terbinafine tested in the present study ranged from 1.0 to 100.0 mg/ml. At this point, it must be
Drug Chem Toxicol, Early Online: 1–6
mentioned that the concentration of 1 mg/ml, the lower tested in the present study, represent the peak plasma concentration of this drug following oral administration in humans (Kovarik et al., 1995). The cultures were treated with terbinafine at concentrations 1.0 mg/ml, 5.0 mg/ml, 25.0 mg/ml, 50.0 mg/ml and 100.0 mg/ml. An untreated culture, a solvent control (DMSO, 10.0 ml/ml and 20.0 ml/ml), a positive control (mitomycin C, 0.1 mg/ml) and five cultures treated with different concentrations of terbinafine were applied to each donor. All cultures were maintained under identical conditions. Chromosomal aberrations and sister chromatid exchanges tests For CA and SCE studies, metaphase preparations were obtained as described by Moorhead et al.(1960) with modifications. For SCE test, the culture medium was supplemented with 10 mg/ml BrdU and cultures were wrapped in aluminum foil to avoid exposure to light. Terbinafine was added 48 h after the initiation of culture. Colchicine (4.105 M) was added 2 h before the collection of the cultures to obtain metaphases. The cultures were treated with a hypotonic solution (0.075 M KCI) for 20 min at 37 C and then fixed twice with cold methanol:acetic acid (3:1, v/v). Three drops of the fixed cell suspension were dropped on clean and cold slides which were maintained at 21 C for 3 days. For CA test, slides were incubated in 2 x SSC (0.3 M sodium chloride and 0.03 M trisodium citrate) solution at 60 C for 5 min and stained with 2% Giemsa solution prepared in a So¨rensen buffer (pH 6.8) for 5 min, washed in distilled water and dried at room temperature. For SCE test, the slides were stained according to fluorescence plus Giemsa technique (Korenberg & Freedlender, 1974), with modifications. In the two tests, all slides were coded prior to scoring and analyzed by a single cytogeneticist. In the CA assay, the frequency of the cells with structural and numeric CA was scored in 100 well-spread metaphases, containing 46 ± 1 chromosomes, per culture, for each treatment and donor (a total of 400 metaphases). Aberrations were classified according to the recommendations of the International System for Human Cytogenetic Nomenclature (ISCN, 2013), as follows: chromatid and chromosome breaks, acentric chromosome fragments, chromatid and chromosome exchanges. Achromatic lesions smaller than the width of a chromatid and continuous with the chromosome axis were recorded into: chromatid and chromosome gaps, albeit not considered as CA. Among the numeric CA observed, only polyploidy was taken into account. The number of each aberration type was summarized for each treatment and the count of cells with CA and the number of CA per cell (CA/cell) were expressed as the mean number ± standard error (SE). The mitotic index (MI) was calculated as the proportion of metaphases in 1000 cells analyzed per culture, for each treatment and donor (a total of 4000 cells) and expressed as mean percentage ± SE. In the SCE test, 25 well-spread second division metaphases, all with 46 chromosomes, were analyzed per culture, for each treatment and donor (a total of 50 metaphases). SCE count was performed according to Lambert et al. (1976), but centromeric exchanges were not considered. The results for
Genotoxic potential of terbinafine
DOI: 10.3109/01480545.2014.959174
Statistical analysis and interpretation of the results
each treatment were expressed as the mean number of SCE per cell (SCE/cell) ± SE. Further, for the determination of the replication index (RI), the proportion of first (M1), second (M2) and third (M3) division cells was scored in 100 consecutive metaphases per culture, for each treatment and donor (a total of 200 cells) and was calculated by the formula: RI ¼ M1+2M2+3M3/100, and expressed as the mean number ± SE.
Statistical analysis was performed by Kruskal-Wallis test, comparing the experimental rates of the tested concentrations of terbinafine and the solvent control obtained in CA, SCE and CBMN tests. Dose–response relationships were determined from the correlation coefficients (r), considered as strong to perfect correlation the following values: 0.75 5 r 51.00 or 1 5 r 5 0.75 (perfect if r ¼ 1 or r ¼ 1) (Vieira, 2008). For all analyses, a significance level of 5% (a ¼ 0.05) was used. For interpretation of results beyond the statistical analysis, complementary criteria were also considered as a positive response: an induction of 45% of cells with CA (Parry et al., 2010), for CA test, and an increase in the frequency of SCE in tested concentrations to two times when compared to the solvent control (Varella-Garcia, 1991), for SCE test. In CBMN assay, the MN, NP and NB rates suggested by Fenech (2007) were considered as indicative of spontaneous induction.
Cytokinesis-block micronucleus test
Drug and Chemical Toxicology Downloaded from informahealthcare.com by York University Libraries on 03/04/15 For personal use only.
3
The CBMN test was carried and analyzed as described by Fenech (2007), with modifications. Terbinafine was added 24 h after the initiation of culture. Cytochalasin B (6 mg/ml) was added 28 h before the collection of the cultures to arrest cytokinesis and obtain binucleated (BN) cells. The cultures were treated with hypotonic solution (0.075 M KCl) at 4 C for 5 min and then fixed twice with cold methanol:acetic acid (3:1, v/v). In the last fixative, 1% formaldehyde was added to preserve the cytoplasm. Slides were prepared by dropping three drops of the fixed cell suspension on clean and cold slides and then air-dried. The slides were incubated at 21 C for 3 days and stained with 5% Giemsa solution prepared in a So¨rensen buffer (pH 6.8), for 5 min, washed in distilled water and dried at room temperature. All slides were coded prior to scoring and analyzed by a single cytogeneticist. Frequency of micronuclei (MN), nucleoplasmic bridges (NP) and nuclear buds (NB) were scored in the CBMN assay. A total of 1000 BN lymphocytes were analyzed per culture, for each treatment and donor (a total of 2000 cells) to determine the number of MN, NP and NB. Total number of MN, NP and NB scored were summarized for each treatment and the count of BN cells with MN and NB was expressed as the mean number ± SE. For determining cytokinesis-block proliferation index (CBPI), 500 cells with well-preserved cytoplasm, containing 1–4 nuclei, were scored per culture, for each treatment and donor. CBPI was calculated according to Surralle´s et al. (1995) using the following formula: N1 þ2N2 þ3ðN3 þN4 Þ CBPI ¼ N
Results Five different concentrations (1.0 mg/ml, 5.0 mg/ml, 25.0 mg/ml, 50.0 mg/ml and 100.0 mg/ml) and eight different parameters (CA, SCE, MN, NP, NB, MI, RI and CBPI) were evaluated to determine the genotoxic and cytotoxic/cytostatic effects of terbinafine in cultured human peripheral lymphocytes. The results of CA test are shown in Table 1.Three types of structural CA were observed for terbinafine in tested concentrations: chromosome and chromatid breaks and acentric chromosome fragments, giving a predominance of chromatid breaks. Other structural aberrations as dicentric and ring chromosomes and chromosomal rearrangements were not observed in any of the tested concentrations of terbinafine. Polyploidy was observed. Among the tested concentrations of terbinafine and solvent control, no significant differences regarding the numbers the cells with CA (p ¼ 0.1943), CA/cell (p ¼ 0.2065) and percentages of MI (p ¼ 0.8003) were reported. Further, the number of cells with CA was not increased in a dose-dependent manner (r ¼ 0.69). No more than 5% of cells with CA, in each one of tested concentrations of terbinafine, were observed, the maximum rate was 4.0 ± 1.29% (100.0 mg/ml). The effects of terbinafine on the SCE and RI are summarized in Table 2. There was no significant difference
where, N1–N4 represent the number of cells with 1–4 nuclei, respectively, and N is the total number of cells scored and expressed as the mean number ± SE.
Table 1. Chromosomal aberrations and mitotic index in human lymphocytes treated with terbinafine in vitro. Treatment Test substance Untreated culture Solvent control Positive control (mitomycin C) Terbinafine
Chromosomal aberrations
Period (h)
Dose
csb
ctb
f
cte
p
CCA (mean* ± SE)
CA/cell (mean ± SE)
MI(%) (mean ± SE)
– 48 48 48
– 20.0 ml/ml 0.1mg/ml 1.0 mg/ml 5.0 mg/ml 25.0 mg/ml 50.0 mg/ml 100.0 mg/ml
3 1 165 0 2 8 3 4
2 10 83 2 6 5 4 11
0 0 9 1 1 1 2 1
0 0 18 0 0 0 0 0
0 0 0 2 0 1 0 2
1.25 ± 0.48 2.50 ± 1.55 38.75 ± 14.48 1.25 ± 0.63 2.25 ± 0.25 3.75 ± 1.18 2.25 ± 0.95 4.00 ± 1.29
0.0125 ± 0.005 0.0275 ± 0.018 0.6875 ± 0.340 0.0125 ± 0.006 0.0225 ± 0.002 0.0375 ± 0.012 0.0225 ± 0.009 0.0450 ± 0.017
3.525 ± 0.14 2.750 ± 0.53 0.875 ± 0.24 2.700 ± 0.38 2.575 ± 0.53 2.325 ± 0.33 2.500 ± 0.33 2.125 ± 0.26
csb, chromosome break; ctb, chromatid break; f, acentric chromosome fragment; cte, chromatid exchange; p, polyploidy; CCA, cells with chromosomal aberrations; CA/cell, chromosomal aberrations per cell; MI, mitotic index. Four hundred metaphases were scored for each treatment to analyze chromosomal aberrations and 4000 lymphocytes were scored for each dose level for the MI. *The average sample rate coincides with CCA percentage.
4
D. Tolomeotti et al.
Drug Chem Toxicol, Early Online: 1–6
Table 2. Sister chromatid exchanges and replication index in human lymphocytes treated with terbinafine in vitro. Test substance
Treatment Period (h)
Untreated culture Solvent control Positive control (mitomycin C) Terbinafine
Min-max
SCE/cell (mean ± SE)
RI (mean ± SE)
1–10 1–15 6–41 0–14 1–14 2–18 2–14 1–22
5.52 ± 0.36 6.32 ± 1.92 24.42 ± 4.06 6.60 ± 2.12 6.14 ± 0.98 6.64 ± 1.32 6.66 ± 0.46 6.68 ± 1.36
2.435 ± 0.055 2.195 ± 0.165 1.745 ± 0.015 2.265 ± 0.135 2.210 ± 0.240 2.145 ± 0.085 2.355 ± 0.025 1.995 ± 0.085
Dose
– 48 48 48
– 20.0 ml/ml 0.1 mg/ml 1.0 mg/ml 5.0 mg/ml 25.0 mg/ml 50.0 mg/ml 100.0 mg/ml
Drug and Chemical Toxicology Downloaded from informahealthcare.com by York University Libraries on 03/04/15 For personal use only.
SCE/cell, sister chromatid exchanges per cell; RI, replication index. Fifty metaphases were scored for each dose level in the SCE test. Two hundred metaphases were scored in each dose level for RI.
Table 3. Micronuclei, nuclear buds and cytokinesis-block proliferation index (CBPI) in human lymphocytes treated with terbinafine in vitro. Treatment Test substance Negative control Solvent control Positive control Terbinafine
Period (h)
Concentration
BN scored
MN per 1000 BN (mean ± SE)
Total number of MN
BNNB (mean ± SE)
Total number of buds
CBPI (mean ± SE)
24 24 24 24 24 24 24 24
– 10 mL/mL 0.1 mg/mL 1 mg/mL 5 mg/mL 25 mg/mL 50 mg/mL 100 mg/mL
2000 2000 2000 2000 2000 2000 2000 2000
6.5 ± 0.50 6.0 ± 2.01 96.5 ± 18.56 7.5 ± 0.50 8.5 ± 0.50 10.0 ± 1.00 10.5 ± 0.50 11.5 ± 1.50
13 12 193 15 17 20 21 23
2.0 ± 0 3.5 ± 0.50 10.0 ± 0 3.0 ± 1.003 4.0 ± 0 1.0 ± 1.003 4.5 ± 0.50 5.5 ± 0.50
4 7 20 6 8 2 9 11
2.01 ± 0.01 2.01 ± 0.01 1.9 ± 0.21 2.0 ± 0.13 2.0 ± 0.21 1.9 ± 0.06 1.7 ± 0.12 1.43 ± 0.18
BN: binucleated cells; MN: micronuclei; BNNB: binucleated cells with nuclear buds; NB: nuclear buds; CBPI: cytokinesis-block proliferation index. Positive control: mitomicyn C.
in the number of SCE/cell (p ¼ 0.9946) and RI (p ¼ 0.5945) among the tested concentrations of terbinafine and solvent control. The number of SCE/cell did not increase (r ¼ 0.54) neither RI decrease (r ¼ 0.59) in a dose-dependent manner. No differences were observed regarding the frequency of the SCE/cell when compared to solvent control (6.32 ± 1.92), with maximum rate 6.68 ± 1.36 (100.0 mg/ml). Table 3 shows the results obtained in the CBMN assay. Among the tested concentrations of terbinafine and solvent control, there was no significant difference in the number of BN cells with MN (p ¼ 0,1103) and NB (p ¼ 0,03587). No significant differences were found in CBPI (p ¼ 0,1853) when treatment and solvent control are compared. NP were not observed in any of the tested terbinafine concentrations.
Discussion Terbinafine is an orally and topically active drug with fungicidal activity against a broad spectrum of fungi (Kovaric et al., 1995). Studies showed that terbinafine has anti-angiogenic and universal anti-tumor proliferation properties, which makes it a very attractive agent for cancer therapy (Ho et al., 2004, 2006; Lee et al., 2003). Chien et al. (2012) demonstrated for the first time that terbinafine might be a potential therapeutic tool in the treatment of oral cancer. Genetic toxicology studies play an important role in evaluating health hazards associated with the exposure of humans and living organisms to chemical substances (Ohno et al., 2005). In general, CA, SCE and MN are considered to
be essential markers of genotoxicity in vitro studies (Celik & Eke, 2011). Current study investigates for the first time the genotoxic potential of terbinafine using CA, SCE and CBMN tests in cultured human lymphocytes. Five terbinafine concentrations were evaluated in our study: the peak plasma concentration of terbinafine following oral administration in humans (Kovaric et al., 1995) (1.0 mg/ml) and four additional concentrations higher than the plasma concentration (5.0 mg/ml, 25.0 mg/ml, 50.0 mg/ml and 100.0 mg/ml). No significant genotoxic effects were observed by the five different parameters (CA, SCE, MN, NP and NB) in all terbinafine tested concentrations. Furthermore, the results were also negative in terms of cytotoxicity and cytostasis, since no significant differences were found in MI, RI and CBPI for any terbinafine concentration. The CA assay in cultured cells has been widely used for many years, and it has been proved to be an useful and sensitive test for the detection of genotoxic agents (Rao et al., 2004). The varieties of CA observed in human lymphocytes treated in vitro with terbinafine were observed for fluconazole, another antifungal drug (Yu¨zba¸siog˘lu et al., 2008). Although in our analysis the structural and numeric CA has also been observed, this datum was not relevant. The limit value of cells with CA, based on Parry et al. (2010), was not exceeded in untreated culture, solvent control or any of the terbinafine concentrations. This complementary criterion agrees with statistical results. In positive control, 38.75 ± 14.48% of the cells with CA, were observed. This datum indicates that this criterion is strong enough to obtain the final response in CA test. The frequencies of CA/cell in untreated
Genotoxic potential of terbinafine
Drug and Chemical Toxicology Downloaded from informahealthcare.com by York University Libraries on 03/04/15 For personal use only.
DOI: 10.3109/01480545.2014.959174
culture, solvent control and tested concentrations of terbinafine ranged from 1.25 to 4.5%, which is in conformity with the proposed by Kasˇuba et al. (1995) for spontaneous CA (range 0 to 6.7%). SCE test is a sensitive test for monitoring DNA damage (Pongsavee, 2009) although little is known about its molecular basis. This analysis has been used to investigate the genotoxic effects of several chemical agents, for example, food additives (Yilmaz et al., 2008, 2009; Zengin et al., 2011). SCE occurs in human lymphocyte cultures, with or without induction by physical or chemical agents, and it seems to reflect the DNA repair by homologous recombination (Garcia-Sagredo, 2008). The frequency of the SCE/cell increases after exposure of cells to genotoxic agents, as demonstrated by Aydemir et al. (2005), C¸elik et al. (2010) and Mamur et al. (2010). The frequencies of SCE/cell in untreated culture, solvent control and all terbinafine concentrations ranged from 5.52 ± 0.36–6.68 ± 1.36 SCE/cell, which is in accordance with the basal frequency suggested by Natarajan & Obe (1986), within the 5–10 SCE/cell. Our results showed that terbinafine does not induce SCE in human lymphocytes because, beyond the statistical results, in none of the tested concentration of terbinafine was observed the two-fold increase in SCE over that of the solvent control. In positive control, 24.42 ± 4.06 SCE/cell was observed, approximately four times greater than that in solvent control. This datum demonstrates the validity of this complementary criterion adopted to obtain the final response in the SCE test. The in vitro MN assay is currently being considered as a suitable method for testing the genotoxicity of chemical and pharmaceutical compounds (Fenech, 2000). The MN, NP and NB rates suggested by Fenech (2007), adopted in this study as indicative of spontaneous induction, were not exceeded in untreated culture, solvent control and terbinafine group. This complementary criterion agrees with statistical results. Results indicate that, under current test conditions, terbinafine does not induce chromosome breaks and/or gain or loss in human lymphocytes. Similar results were found for other drugs (Ila & Ilhan, 2012; Kasurka et al., 2011). However, griseofulvin, another antifungal drug, induced MN formation in cultured human lymphocytes and was considered a strong aneuploidogenic agent (Kolachana & Smith, 1994). MI explains the effects of the chemical compounds on the G2 stage of the cell cycle, while RI reflects the effects on the S and G2 stages (Arslan et al., 2008). CBPI estimates the average number of cell division of a cell population and may be considered a cell kinetic index (Surralle´s et al., 1995). Since a significant reduction of MI, RI and CBPI rates in cultures treated with terbinafine was not observed, terbinafine does not seem to produce effects on the proliferation/mitotic index when its concentration is equal to or less than 100.0 mg/ ml. Terbinafine at concentrations 30–60 mM did not inhibit the growth rate of the cultured human gingival fibroblasts, and at doses 10–150 mM it was not cytotoxic for the cultured untransformed human fibroblasts, with no effect on cell proliferation in this culture (Lee et al., 2003). Therefore, the data obtained in current study seem to corroborate the results obtained by Lee et al. (2003).
5
Conclusion The results in the current study show that terbinafine does not induce clastogenesis, DNA effects, aneugenesis, cytotoxicity and cytostasis in human lymphocytes in vitro. Since very few studies have been published on the genotoxicity of this antimicotic drug, our results contribute with data towards a safer use of the drug, already widely used in the treatment of dermatomycoses in humans and with a potential application as an anti-angiogenic and anti-tumor agent.
Acknowledgements We are grateful to Fundac¸a˜o Arauca´ria de Apoio ao Desenvolvimento Cientı´fico e Tecnolo´gico do Parana´ for financial support. Tolomeotti, D. was the recipient of a Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) fellowship. Sant’Anna, J.R. is the recipient of a Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES) fellowship.
Declaration of interest The authors declare that there is no conflict of interest.
References Arslan M, Topaktas M, Rencuzogullari E. (2008). The effects of boric acid on sister chromatid exchanges and chromosome aberrations in cultured human lymphocytes. Cytotechnology, 56:91–96. Aydemir N, C¸elikler S, Bilalog˘lu R. (2005). In vitro genotoxic effects of the anticancer drug gemcitabine in human lymphocytes. Mutat Res, 582:35–41. Baboota S, Al-Azaki A, Kohli K, et al. (2007). Development and evaluation of a microemulsion formulation for transdermal delivery of terbinafine. PDA J Pharmaceut Sci Technol, 61:276–285. Baran R, Sigurgeirsson B, de Berker D, et al. (2007). A multicentre, randomized, controlled study of the efficacy, safety and costeffectiveness of a combination therapy with amorolfine nail lacquer and oral terbinafine compared with oral terbinafine alone for the treatment of onychomycosis with matrix involvement. British J Dermatol, 157:149–157. Bayel I. (2006). Terbinafin’in insan lenfosit ku¨ltu¨rlerindeki etkilerinin karde¸s kromatid degˇi¸simi (sister chromatid exchange) yo¨ntemi ile in ¨ niversitesi vitro ara¸stırılması. (Yu¨ksek Lisans Tezi). Mersin: Mersin U Sag_lik Bilimleri Enstitu¨su¨,61s. Bechter R, Schmid BP. (1987). Teratogenicity in vitro - A comparative study of four antimycotic drugs using the whole-embryo culture system. Toxicol In Vitro, 1:11–15. C¸elik A, Eke D. (2011). The assessment of cytotoxicity and genotoxicity of tetracycline antibiotic in human blood lymphocytes using CBMN and SCE analysis, in vitro. Int J Hum Genet, 11:23–29. Celik M, Aksoy H, Yilmaz S. (2010). Evaluation of beauvericin genotoxicity with the chromosomal aberrations, sister-chromatid exchanges and micronucleus assays. Ecotoxicol Environ. Saf, 73:1553–1557. Chien MH, Lee TS, Kao C, et al. (2012). Terbinafine inhibits oral squamous cell carcinoma growth through anti-cancer cell proliferation and anti-angiogenesis. Mol Carcinog, 51:389–399. Favre B, Ryder NS. (1996). Characterization of squalene epoxidase activity from the dermatophyte Trichophyton rubrum and its inhibition by terbinafine and other antimycotic agents. Antimicrob Agents Chemother, 40:443–447. Fenech M. (2000). A mathematical model of the in vitro micronucleus assay predicts false negative results if micronuclei are not specifically scored in binucleated cells or in cells that have completed one nuclear division. Mutagenesis, 15:329–336. Fenech M. (2007). Cytokinesis-block micronucleus cytome assay. Nat Protoc, 2:1084–1104.
Drug and Chemical Toxicology Downloaded from informahealthcare.com by York University Libraries on 03/04/15 For personal use only.
6
D. Tolomeotti et al.
Garcia-Sagredo JM. (2008). Fifty years of cytogenetics: a parallel view of the evolution of cytogenetics and genotoxicology. Biochim Biophys Acta, 1779:363–375. Gregurek-Novak T. (2004). Effect of infection with Trichophyton mentagrophytes varietas interdigitale on phagocytosis in humans. J European Acad Dermatol Venereol, 18:160–163. Ho PY, Liang YC, Ho YS, et al. (2004). Inhibition of human vascular endothelial cells proliferation by terbinafine. Int J Cancer, 111:51–59. Ho PY, Zhong WB, Ho YS, Lee WS. (2006). Terbinafine inhibits endothelial cell migration through suppression of the Rho-mediated pathway. Mol Cancer Ther, 5:3130–3138. Ila HB, Ilhan A. (2012). In vitro genotoxic perspective of Tamiflu. Cytotechnology, 64:443–449. ISCN (2013). An international system for human cytogenetic nomenclature (2013). In: Shaffer LG, McGowan-Jordan JR, Schmid M, eds. (Karger Medical and Scientific Publishers). Kasˇuba V, Sˇentija K, Garaj-Vrhovac V, Fucˇic´ A. (1995). Chromosome aberrations in peripheral blood lymphocytes from control individuals. Mutat Res, 346:187–193. Kasurka CB, S ¸ ekerog˘lu ZA, S ¸ ekerog˘lu V. (2011). Evaluation of the genotoxicity and cytotoxicity of fexofenadine in cultured human peripheral blood lymphocytes. Toxicol In Vitro, 25:1480–1484. Kolachana P, Smith MT. (1994). Induction of kinetochore-positive micronuclei in human lymphocytes by the anti-fungal drug griseofulvin. Mutat Res, 322:151–159. Korenberg JR, Freedlender EF. (1974). Giemsa technique for the detection of sister chromatid exchanges. Chromosoma, 48:355–360. Kovarik JM, Mueller EA, Zehender H, et al. (1995). Multiple-dose pharmacokinetics and distribution in tissue of terbinafine and metabolites. Antimicrob Agents Chemother, 39:2738–2741. Lambert BO, Hansson K, Lindsten J, et al. (1976). Bromodeoxyuridineinduced sister chromatid exchanges in human lymphocytes. Hereditas, 83:163–174. Lee WS, Chen RJ, Wang YJ, et al. (2003). In vitro and in vivo studies of the anticancer action of terbinafine in human cancer cell lines: G0/G1 p53-associated cell cycle arrest. Int J Cancer, 106:125–137. ¨ nal F, Yilmaz S. (2010). Does potassium Mamur S, Yu¨zba¸siog˘lu D, U sorbate induce genotoxic or mutagenic effects in lymphocytes? Toxicol In Vitro, 24:790–794 Mehta K, Bhatt DC. (2011). Preparation, optimization and in vitro microbiological efficacy of antifungal microemulsion. Int J Pharmaceut Sci Res, 2:2424–2429. Moorhead PS, Nowell PC, Mellman WJ, et al. (1960). Chromosome preparations of leukocytes cultured from human peripheral blood. Exp Cell Res 1960, 20:613–616.
Drug Chem Toxicol, Early Online: 1–6
Natarajan AT, Obe G. (1986). How do in vivo mammalian assays compare to in vitro assays in their ability to detect mutagens? Mutat Res, 167:189–201. Newland JG, Abdel-Rahman SM. (2009). Update on terbinafine with a focus on dermatophytoses. Clin Cosmet Investig Dermatol, 2:49–63. Novartis (2013). LamisilÕ (terbinafine hydrochloride). Avaliable at: http://www.acessdata.fda.gov/drugsatfda_docs/label/2013/ 022071s009Ibl.pdf. Ohno K, Tanaka-Azuma Y, Yoneda Y, Yamada T. (2005). Genotoxicity test system based on p53R2 gene expression in human cells: examination with 80 chemicals. Mutat Res, 588:47–57. Parry JM, Parry E, Phrakonkham P, Corvi R. (2010). Analysis of published data for top concentration considerations in mammalian cell genotoxicity testing. Mutagenesis, 25:531–538. Petranyi G, Meingassner JG, Mieth H. (1987). Antifungal activity of the allylamine derivative terbinafine in vitro. Antimicrob Agents Chemother, 31:1365–1368. Pongsavee M. (2009). Effect of borax on immune cell proliferation and sister chromatid exchange in human chromosomes. J Occup Med Toxicol, 4:1–6. Rao KS, Xu Y, Shaw E, Parton JW. (2004). Mutagenicity testing applied for regulation of developing products. Curr Separations, 20:141–144. Sharma V, Bhatia R. (2011). Triazoles in antifungal therapy: A review. Int J Pharm Biomed Res, 2:417–427. Surralle´s J, Xamena N, Creus A, et al. (1995). Induction of micronuclei by five pyrethroid insecticides in whole blood and isolated human lymphocytes cultures. Mutat Res, 341:169–184. Varella-Garcia M. (1991). Teste de trocas entre croma´tides-irma˜s. In: Rabello-Gay MN, Rodrigues MA, Monteleone-Neto R, editors. Mutageˆnese, teratogeˆnese e carcinogeˆnese: me´todos e crite´rios de avaliac¸a˜o. Ribeira˜o Preto, SP: SBG, p. 123–139. Vieira S. (2008). Introduc¸a˜o a` bioestatı´stica. Rio de Janeiro, RJ, Brasil: Elsevier. ¨ nal F, Yu¨zba¸siog˘lu D, Aksoy H. (2008). Clastogenic effects Yilmaz S, U of food additive citric acid in human peripheral lymphocytes. Cytotechnology, 56:137–144. ¨ nal F, Yu¨zba¸siog˘lu D. (2009). The in vitro genotoxicity Yilmaz S, U of benzoic acid in human peripheral blood lymphocytes. Cytotechnology, 60:55–61. ¨ nal F, Yilmaz S, et al. (2008). Genotoxicity testing of Yu¨zba¸siog˘lu D, U fluconazole in vivo and in vitro. Mutat Res, 649:155–160. ¨ nal F, et al. (2011). The evaluation of the Zengin N, Yu¨zba¸siog˘lu D, U genotoxicity of two food preservatives: Sodium benzoate and potassium benzoate. Food Chem Toxicol, 49:763–769.
